The budding of retroviruses is dependent upon small L-domain sequences encoded in their Gag polyproteins that serve as binding sites for cellular proteins involved in endosomal sorting (ESCRT-I, -II, -III). These cell proteins provide the mechanical means for virus-like particles to release from the plasma membrane. Depending upon the retrovirus, three different L domains are used, individually or in combination. To understand the nature of these protein complexes, Gag polyproteins from avian sarcoma (ASV), human immunodeficiency, type I, (HIV-1) and Moloney murine leukemia (MuLV) viruses will be isolated from cells under native conditions, associated cell proteins fractionated by two-dimensional chromatography, and those that bind to Gag in an L-domain dependent fashion will be identified by mass spectrographic techniques. This will define those cellular proteins that are shared or not. By analysis of complexes derived from wild type and Gag containing L-domain deletions but fused to specific ESCRT-I, -II, -III proteins that rescue or not the budding defect, the entry points for each virus into the budding pathway will be elucidated. Both ASV and MuLV Gag use related but different PPxY L-domain motifs, which are specific binding sites for E3 ubiquitin ligases. These motifs will be exchanged between ASV and MuLV Gag polyproteins to demonstrate that this changes the specificity for the E3 protein used in the budding process. Site directed mutagenesis of residues that differ between related PY motifs and siRNA phenotypic depletion inside of cells targeting different E3 proteins will also be carried out to establish their mechanistic role in the budding process. The possible function of the C2 transport domain of the E3 ubiquitin ligase, Nedd4, will be examined for a role in transport of ASV Gag to the cell surface. Finally, a series of known ubiquitin mutants will be analyzed for their effect on Gag release. Taken together, these experiments will provide important new information about the mechanism of budding and suggest cellular targets for the development of antiviral agents that disrupt the viral budding process. Retroviruses may be less likely to develop drug resistance to such agents than to drugs that target viral encoded enzymes. PUBLIC HEALTH RELEVANCE: The mechanism of budding from cells of three different retroviruses will be compared using biochemical proteomics and site directed mutagenesis techniques. These studies will lay the basic foundation for understanding an essential process in viral replication and suggest targets for the development of antiviral agents for which retroviruses may be less likely to develop drug resistance variants.